2016 Volume 39 Issue 5 Pages 883-886
The urinary metabolic profiles of three hallucinogenic 2,5-dimethoxy-4-alkylthiophenethylamine analogs: 2,5-dimethoxy-4-ethylthiophenethylamine (2C-T-2), 2,5-dimethoxy-4-isopropylthiophenethylamine (2C-T-4), and 2,5-dimethoxy-4-propylthiophenethylamine (2C-T-7), were investigated in rats. For each drug, four male Sprague-Dawley rats were orally administered 10 mg/kg of 2C-T-2, 2C-T-4, or 2C-T-7, and urine was collected 0–24 and 24–48 h after administration. The urine samples were processed by liquid–liquid extraction, and the extracts were analyzed by liquid chromatography/mass spectrometry to quantify the metabolites. The metabolic patterns of these drugs were different: for 2C-T-7, the principal metabolite was the β-hydroxylated-N-acetylated-sulfoxide, whereas for 2C-T-2 and 2C-T-4 the major metabolites were the N-acetylated-sulfoxide and S-methylated-N-acetylated-sulfoxide, respectively.
2,5-Dimethoxy-4-alkylthiophenethylamine analogs (2C-T analogs) are phenethylamine hallucinogenic drugs first synthesized by Shulgin and Shulgin.1) Shulgin synthesized more than ten psychoactive 2C-T analogs, and among them, 2,5-dimethoxy-4-ethylthiophenethylamine (2C-T-2), 2,5-dimethoxy-4-isopropylthiophenethylamine (2C-T-4), and 2,5-dimethoxy-4-propylthiophenethylamine (2C-T-7) (Fig. 1) have been widely abused since the early 2000 s.2) These drugs are now under legislative control in many countries, including Japan.
Some reports of the metabolism of 2C-T analogs have been published.3–7) 2C-T analogs are metabolized by sulfur oxidation, N-acetylation, oxidation of the S-alkyl group, S-dealkylation followed by S-methylation, and deamination followed by oxidation to form a carboxylic acid (Fig. 1). However, in-depth pharmacokinetic analysis of 2C-T analogs has never been performed. In the present study, we report the quantitative analysis of the metabolites of 2C-T-2, 2C-T-4, and 2C-T-7 in rat urine to clarify the metabolic behavior of these compounds.
Authentic standards of 2C-T-2, 2C-T-4, 2C-T-7 and their metabolites were synthesized in our laboratory as described previously.3,4,8) β-Glucuronidase/aryl sulfatase (from Helix pomatia; β-glucuronidase, 5.2 units/mL; aryl sulfatase, 2.08 units/mL) was purchased from Calbiochem-Novabiochem Co., Ltd. (La Jolla, CA, U.S.A.). All other chemicals used were of analytical grade.
Drug Administration and Urine SamplingFor each drug, four male Sprague-Dawley rats (seven week old) were orally administered 10 mg/kg of 2C-T-2 hydrochloride, 2C-T-4 hydrochloride, or 2C-T-7 hydrochloride and placed in metabolic cages. The 0–24 and 24–48 h urinary fractions were collected and stored at −20°C. All animal experiments were approved by the Animal Ethics Committee of the National Research Institute of Police Science.
Extraction of the Metabolites and Sample PreparationWater (90 µL) and 0.5 M acetate buffer (50 µL, pH 5.0) containing β-glucuronidase/aryl sulfatase (β-glucuronidase, 5.2×10−3 units) were added to the urine sample (10 µL) and the mixture was incubated at 60°C for 90 min to hydrolyze the conjugate. Next, 0.5 M sodium borate buffer (0.4 mL, pH 9.0) was added to the hydrolyzed urine sample and the mixture was extracted with chloroform–2-propanol (3 : 1, 3×1 mL, basic fraction). To the combined organic layer, glycerol–water (1 : 1, 20 µL) was added and evaporated under a gentle stream of nitrogen. The residue was dissolved in 10 mmol/L ammonium acetate (pH 5.0)–methanol (4 : 1, 200 µL) and the solution (5 µL) was injected into the liquid chromatography/mass spectrometry (LC/MS) system.
Hydrochloric acid (0.1 M, 0.4 mL) was added to another hydrolyzed urine sample and extracted with diethyl ether (3×1 mL, acidic fraction). Glycerol–water (1 : 1, 20 µL) was added to the combined organic layer and evaporated under a gentle stream of nitrogen. The residue was dissolved in 0.1% acetic acid–methanol (4 : 1, 200 µL) and the solution (5 µL) was injected into the LC/MS system.
The recoveries of the drugs and their metabolites ranged from 72.0 to 120.3% (n=4) and the coefficients of variation for all compounds were below 15%.
Liquid Chromatography/Mass SpectrometryLC/MS analysis was performed with a liquid chromatograph (NanoSpace SI-2; Shiseido, Tokyo, Japan) connected to a mass spectrometer (TSQ Quantum; Thermo Scientific, Waltham, MA, U.S.A.). The conditions were as follows: column, Sunfire C18 (2.1×150 mm, particle diameter 3.5 µm; Waters Corporation, Milford, MA, U.S.A.) maintained at 40°C; mobile phase composition, (for basic fraction) 10 mmol/L ammonium acetate (pH 5.0) (A) and methanol (B); linear gradient mode, 5% B to 60% B over 15 min, 60% B (6 min hold), returning to 5% B in 1 min; (for acidic fraction) 0.1% acetic acid (A) and methanol (B); linear gradient mode, 20% B to 80% B over 15 min, 80% B (6 min hold), returning to 20% B in 1 min; flow rate, 0.3 mL/min; mass spectrometry interface, positive electrospray ionization; analysis mode, selected reaction monitoring (SRM). The SRM transitions and collision energies for each compound are listed in Table 1.
| Compound | Precursor ion (m/z) | Monitoring ion (m/z) | Collision energy (eV) | Percent of the primed dose of drug (Mean±S.D.) | |
|---|---|---|---|---|---|
| 0–24 h | 24–48 h | ||||
| <2C-T-2> | |||||
| 2C-T-2 | 242 | 225 | 11 | Trace | Trace |
| 2C-T-2-SO | 258 | 213 | 16 | 2.6±0.33 | 0.12±0.037 |
| 2C-T-2-SO2 | 274 | 181 | 18 | 0.20±0.013 | Trace |
| 2C-T-2-SO-Ac | 300 | 213 | 21 | 19.9±4.4 | 1.2±0.32 |
| 2C-T-2-SO2-Ac | 316 | 257 | 18 | 12.8±1.3 | 0.76±0.20 |
| β-OH-2C-T-2 | 258 | 241 | 13 | ND | ND |
| β-OH-2C-T-2-SO | 274 | 213 | 17 | ND | ND |
| β-OH-2C-T-2-SO2 | 290 | 273 | 12 | ND | ND |
| β-OH-2C-T-2-SO-Ac | 316 | 164 | 18 | 0.75±0.014 | Trace |
| β-OH-2C-T-2-SO2-Ac | 332 | 164 | 24 | 0.52±0.041 | Trace |
| S-Me | 228 | 211 | 11 | 0.14±0.031 | Trace |
| S-Me-SO | 244 | 151 | 20 | 0.090±0.017 | ND |
| S-Me-SO2 | 260 | 243 | 12 | 0.066±0.024 | ND |
| S-Me-SO-Ac | 286 | 227 | 17 | 3.5±0.48 | 0.20±0.056 |
| S-Me-SO2-Ac | 302 | 243 | 19 | 1.3±0.090 | 0.087±0.029 |
| 2C-T-2-CBA | 257 | 211 | 11 | 0.65±0.16 | Trace |
| 2C-T-2-CBA-SO | 273 | 244 | 18 | 3.8±0.20 | 0.22±0.070 |
| 2C-T-2-CBA-SO2 | 289 | 196 | 16 | 0.41±0.056 | Trace |
| <2C-T-4> | |||||
| 2C-T-4 | 256 | 197 | 19 | Trace | ND |
| 2C-T-4-SO | 272 | 213 | 15 | 1.6±0.24 | 0.077±0.037 |
| 2C-T-4-SO2 | 288 | 181 | 20 | 0.097±0.014 | Trace |
| 2C-T-4-SO-Ac | 314 | 195 | 16 | 3.3±0.72 | 0.17±0.048 |
| 2C-T-4-SO2-Ac | 330 | 181 | 26 | 1.9±0.32 | 0.10±0.022 |
| β-OH-2C-T-4 | 272 | 255 | 11 | ND | ND |
| β-OH-2C-T-4-SO | 288 | 199 | 18 | ND | ND |
| β-OH-2C-T-4-SO2 | 304 | 150 | 23 | ND | ND |
| β-OH-2C-T-4-SO-Ac | 330 | 211 | 19 | Trace | ND |
| β-OH-2C-T-4-SO2-Ac | 346 | 164 | 25 | ND | ND |
| S-Me | See above | 0.59±0.21 | Trace | ||
| S-Me-SO | 0.38±0.070 | Trace | |||
| S-Me-SO2 | ND | ND | |||
| S-Me-SO-Ac | 13.4±1.2 | 0.86±0.38 | |||
| S-Me-SO2-Ac | 5.8±0.50 | 0.41±0.17 | |||
| 2C-T-4-CBA | 271 | 183 | 15 | ND | ND |
| 2C-T-4-CBA-SO | 287 | 181 | 18 | 1.2±0.45 | 0.068±0.032 |
| 2C-T-4-CBA-SO2 | 303 | 243 | 13 | 0.27±0.13 | Trace |
| <2C-T-7> | |||||
| 2C-T-7 | 256 | 239 | 12 | Trace | ND |
| 2C-T-7-SO | 272 | 213 | 17 | 2.0±0.64 | Trace |
| 2C-T-7-SO2 | Not determined | ||||
| 2C-T-7-SO-Ac | 314 | 149 | 28 | 1.1±0.23 | Trace |
| 2C-T-7-SO2-Ac | 330 | 271 | 19 | 0.18±0.040 | ND |
| β-OH-2C-T-7 | 272 | 255 | 12 | 0.96±0.33 | Trace |
| β-OH-2C-T-7-SO | 288 | 199 | 17 | 0.34±0.047 | ND |
| β-OH-2C-T-7-SO2 | Not determined | ||||
| β-OH-2C-T-7-SO-Ac | 330 | 211 | 19 | 18.5±3.1 | 0.43±0.13 |
| β-OH-2C-T-7-SO2-Ac | 346 | 164 | 22 | 7.5±1.1 | 0.20±0.067 |
| γ-OH-2C-T-7 | 272 | 255 | 12 | 0.082±0.038 | ND |
| γ-OH-2C-T-7-SO | 288 | 213 | 17 | Trace | ND |
| γ-OH-2C-T-7-SO2 | Not determined | ||||
| γ-OH-2C-T-7-SO-Ac | 330 | 312 | 14 | 0.33±0.052 | ND |
| γ-OH-2C-T-7-SO2-Ac | 346 | 164 | 23 | 0.23±0.041 | ND |
| S-Me | See above | Trace | Trace | ||
| S-Me-SO | Trace | ND | |||
| S-Me-SO2 | ND | ND | |||
| S-Me-SO-Ac | 1.7±0.36 | Trace | |||
| S-Me-SO2-Ac | 0.78±0.092 | Trace | |||
| 2C-T-7-CBA | 271 | 183 | 15 | Trace | ND |
| 2C-T-7-CBA-SO | 287 | 181 | 18 | 2.2±0.78 | Trace |
| 2C-T-7-CBA-SO2 | 303 | 196 | 16 | 0.19±0.056 | ND |
| <IS> | |||||
| 2,5-Dimethoxyphenylacetic acid | 197 | 151 | 10 | ||
| Diphenhydramine | 256 | 167 | 14 | ||
S.D., standard deviation; ND, not detected; SO, sulfoxide; SO2, sulfone; Ac, N-acetylated; β-OH, β-hydroxylated; γ-OH, γ-hydroxylated; CBA, carboxylic acid; S-Me, S-methylated.
Authentic standards of 2C-T-2, 2C-T-4, 2C-T-7, and their metabolites were added to the control rat urine and processed as described above to obtain the calibration curves. Excellent linearity was obtained over the concentration range 0.1–40 µg/mL with a correlation coefficient of 0.99.
The excretory profiles of 2C-T-2, 2C-T-4, and 2C-T-7 in rat urine are summarized in Table 1. The main 2C-T-2 metabolite excreted in rat urine was N-acetylated-sulfoxide metabolite (2C-T-2-SO-Ac), accounting for 21.1% of the dose at 48 h. The amounts of the other metabolites (>1%) were 2C-T-2-SO2-Ac (13.6%), 2C-T-2-CBA-SO (4.0%), S-Me-SO-Ac (3.7%), 2C-T-2-SO (2.7%), and S-Me-SO2-Ac (1.4%) (SO2, sulfone; CBA, carboxylic acid; S-Me, S-methylated). β-Hydroxylated metabolites (β-OH-2C-T-2-SO-Ac and β-OH-2C-T-2-SO2-Ac) were excreted as minor metabolites. In contrast to 2C-T-2, the main metabolite of 2C-T-4 was the S-methylated-N-acetylated-sulfoxide (S-Me-SO-Ac), accounting for 14.3% of the dose at 48 h. The amounts of the other metabolites (>1%) were S-Me-SO2-Ac (6.2%), 2C-T-4-SO-Ac (3.5%), 2C-T-4-SO2-Ac (2.0%), 2C-T-4-SO (1.7%), and 2C-T-4-CBA-SO (1.3%). Almost no β-hydroxylated metabolite was detected in urine. In contrast, the main metabolite of 2C-T-7 in rat urine was the β-hydroxylated-N-acetylated-sulfoxide (β-OH-2C-T-7-SO-Ac), accounting for 18.9% of the dose at 48 h. The amounts of the other metabolites (>1%) were β-OH-2C-T-7-SO2-Ac (7.7%), 2C-T-7-CBA-SO (2.2%), 2C-T-7-SO (2.0%), S-Me-SO-Ac (1.7%), and 2C-T-7-SO-Ac (1.1%). In all cases, only traces of the parent drugs were detected in the urine sample.
The metabolic patterns of three 2C-T analogs were different from each other, even though these compounds have similar chemical structures. The main metabolites of 2C-T-2, 2C-T-4, and 2C-T-7 were the N-acetylated-sulfoxide (2C-T-2-SO-Ac), the S-methylated-N-acetylated-sulfoxide (S-Me-SO-Ac), and the β-hydroxylated-N-acetylated-sulfoxide (β-OH-2C-T-7-SO-Ac), respectively. In the case of 2C-T-7, the amount of β-hydroxylated metabolites excreted into rat urine was much larger than that of γ-hydroxylated metabolites, indicating that the S-propyl group of 2C-T-7 tends to undergo ω-1-hydroxylation, rather than ω-hydroxylation. On the other hand, α-hydroxylation of S-alkyl group resulted in S-dealkylation, followed by S-methylation. In particular, S-isopropyl group in 2C-T-4 was susceptible to this reaction. To our knowledge, these are the first detailed data on the in vivo metabolism of 2C-T analogs containing S-alkyl group on the aromatic ring.
The metabolism of 2C-T analogs in human is still unclear. Clarification of species differences in the metabolism of 2C-T analogs is an issue to be solved in the future.
The authors declare no conflict of interest.
